Abstract Amelogenesis is a biological process by which highly specialized enamel organ epithelial cells called ameloblasts transport substantial amounts of calcium ions and enamel proteins into the secretory enamel matrix and manufacture a biomaterial of exceptional structural and mechanical properties: tooth enamel (Pandya and Diekwisch 2018). Recent studies have identified some of the calcium channels in dental enamel cells at the basal ameloblast aspect (Nurbaeva et al. 2015, Lacruz 2017). However, there is remarkably little agreement on the mechanisms of ion and protein trafficking at the secretory ameloblast pole and throughout the ameloblast cell body. In support of the present application we have developed three highly innovative models that will address knowledge gaps and advance our understanding of physiological and pathological amelogenesis, including (i) a conditional clathrin deletion mouse model, (ii) an ameloblast 3D bioreactor cell culture model, and (iii) a new liquid cell atomic resolution imaging technology for life in situ imaging of vesicular and extracellular enamel matrices. Establishment of a clathrin knockout model represents significant progress in the area of vesicular trafficking research and a powerful tool to study the function of coated vesicles during amelogenesis. Clathrin-coated vesicles are among the most abundant cellular vesicles, and loss of clathrin has been associated with severe and usually lethal phenotypes (Robinson 2015). Here we present exciting preliminary data demonstrating that clathrin depletion during amelogenesis resulted in altered enamel prism structure and crystal density. Our 3D bioreactor amelogenesis model marks another milestone in enamel research as it promoted the propagation of elongated amelogenin secreting cells, overcoming shortcomings of traditional 2D ameloblast cell culture technology. Third, our atomic resolution liquid chamber model facilitates unprecedented in situ imaging of vesicular contents and native enamel matrix, allowing for the identification of matrix/mineral clusters at the earliest stages of amelogenesis. In response to RFA-DE- 19-004 we have now designed a research plan to develop and optimize these model systems (UG3 phase) and to validate their physiological relevance and usefulness for understanding mechanisms of enamel development and disease during the UH3 phase.